Modified bacteriophage

ABSTRACT

A modified bacteriophage capable of infecting a plurality of different target bacteria, which bacteriophage includes a toxin gene encoding a toxin protein which is toxic to the target bacteria; wherein the bacteriophage is lytic; and wherein the bacteriophage expresses host range determinant proteins which have a plurality of bacterial host specificities.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. application Ser. No. 16/092,096filed Oct. 8, 2018, now U.S. Pat. No. 11,732,243, which is a 35 U.S.C.371 U.S. National Phase Application based on International ApplicationNo. PCT/EP2017/058470 filed Apr. 7, 2017 (published as WO2017/174810 onOct. 12, 2017) which claims the benefit of United Kingdom PatentApplication 1606319.0 filed Apr. 8, 2016, each of which is herebyincorporated by reference in its entirety.

The present invention relates to a modified bacteriophage, uses thereof,and compositions containing the modified bacteriophage.

SEQUENCE LISTING DISCLOSURE

This application includes as part of its disclosure a biologicalsequence listing which is a being concurrently submitted throughEFS-Web. Said biological sequence listing is contained in a file named“1143297o003602.txt” which was created on May 10, 2023, and has a sizeof 86,079 bytes, and is hereby incorporated by reference in itsentirety.

BACKGROUND TO THE INVENTION

The World Health Organisation's 2014 report on global surveillance ofantimicrobial resistance reveals that antibiotic resistance is a globalproblem that is jeopardising the ability to treat common infections inthe community and hospitals. Without urgent action, the world is headingtowards a post-antibiotic era, in which common infections and minorinjuries, which have been treatable for decades, can once again kill(WHO, 2014). Antibiotic resistance complicates patients' recovery fromeven minor operations and is increasingly causing treatment failures. Infact, there are now strains of some genera of bacteria circulatingglobally which are resistant to all available antibiotics. Such strainscommonly fall within the scope of the so-called ESKAPEpathogens—Enterococcus faecium, Staphylococcus aureus, Klebsiellapneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa andEnterobacter species (Boucher et al., 2009). The term ESKAPE pathogenswas coined by Boucher et al., to emphasize that these bacteria currentlycause a majority of hospital infections in the US and Europe and caneffectively “escape” the majority, if not all, available antibioticswith panantibiotic-resistant infections now occurring. The death ratefor patients with serious infections caused by common bacteria treatedin hospitals is approximately twice that of patients with infectionscaused by the same non-resistant bacteria, e.g. people withmethicillin-resistant Staphylococcus aureus (MRSA) infections areestimated to be 64% more likely to die than people with a non-resistantform of the infection (WHO, 2014). Of the Gram positive bacteria,methicillin resistant S. aureus continues to be a major cause ofmorbidity and mortality in hospitals in the US and Europe. However, inmore recent years, several highly resistant Gram negative pathogens,including Acinetobacter species, multidrug resistant (MDR) P.aeruginosa, and carbapenem-resistant Klebsiella species and Escherichiacoli, have emerged as major pathogens causing serious, and sometimesuntreatable, infections. Advances in medicine mean that increasinglycomplex procedures take place: and these advances are leading to agrowing number of elderly patients and patients undergoing surgery,transplantation, and chemotherapy all of which will produce an evengreater number of immunocompromised individuals at risk of theseinfections (Walker et al., 2009). This phenomenon has led to a greaterdependence on, and requirement for, effective antibiotics.

P. aeruginosa is one bacterium which is frequently multi-drug resistant(MDR) having intrinsic resistance due to low permeability of its outermembrane limiting drugs getting into the cell, and a multitude of effluxpumps to expel any drugs that successfully manage to enter the cell. P.aeruginosa is also acquiring additional resistance mechanisms, includingresistance to the “antibiotics of last resort” for Gram negatives, thecarbapenems. P. aeruginosa causes approximately 10% of all hospitalacquired infections and is the second leading cause of hospital-acquiredpneumonia, which accounts for 50% of all hospital-acquired infectionprescribing. P. aeruginosa infections in hospitals commonly requireintravenous (IV) treatment with current standard of care for P.aeruginosa infections dictating that patients are treated with at leasttwo antibiotics. Unfortunately, resistance frequently develops inpatients during therapy. With so few new classes of antibiotic developedand approved for market within the last 30-40 years, there is a criticalneed for novel, safe and effective antibacterial agents.

As an alternative to conventional antibiotics, one family of proteinswhich demonstrate broad spectrum antibacterial activity inside bacteriacomprises the α/β-type small acid-soluble spore proteins (knownhenceforth as SASP). Inside bacteria, SASP bind to the bacterial DNA:visualisation of this process, using cryoelectron microscopy, has shownthat SspC, the most studied SASP, coats the DNA and forms protrudingdomains and modifies the DNA structure (Francesconi et al., 1988;Frenkiel-Krispin et al., 2004) from B-like (pitch 3.4 nm) towards A-like(3.18 nm; A-like DNA has a pitch of 2.8 nm). The protruding SspC motifsinteract with adjacent DNA-SspC filaments packing the filaments into atight assembly of nucleo-protein helices. In 2008, Lee et al. reportedthe crystal structure at 2.1 {acute over (Å)} resolution of an α/β-typeSASP bound to a 10-bp DNA duplex. In the complex, the α/β-type SASPadopt a helix-turn-helix motif, interact with DNA through minor groovecontacts, bind to approximately 6 bp of DNA as a dimer and the DNA is inan A-B type conformation. In this way DNA replication is halted and,where bound, SASP prevent DNA transcription. SASP bind to DNA in anon-sequence specific manner (Nicholson et al., 1990) so that mutationsin the bacterial DNA do not affect the binding of SASP. Sequences ofα/β-type SASP may be found in appendix 1 of WO02/40678, including SASP-Cfrom Bacillus megaterium which is the preferred α/β-type SASP.

WO02/40678 describes the use as an antimicrobial agent of bacteriophagemodified to incorporate a SASP gene. In order to provide effectiveproduction of the modified bacteriophage in a bacterial host, WO02/40678aims to avoid factors which may disrupt proliferation of the productionhost, such as expression of the SASP gene during production. To thisend, the SASP gene was put under the control of an inducible promoter.In a preferred arrangement, at least one of the gene encoding productsinvolved in the lytic process was inactivated.

WO2009019293 describes that effective production of bacteriophage may beachieved where the bacteriophage has been modified to delete a lysisgene and to carry a gene encoding a SASP under the control of a promoterwhich is controlled independently of the bacteriophage, and which isconstitutive with no exogenous or in trans regulation necessary orprovided. Such a modified bacteriophage is maintained as a prophage in amanufacturing host strain, and may be amplified by suitable inductionmethods to synthesise new phage within the manufacturing host strain. Inthis arrangement, the manufacturing host strain must be lysed by theaddition of exogenous substances, e.g. lytic enzymes or chemicals, inorder to release viable phage. An example of a suitable promoter is thefbaA promoter from S. aureus which is used to drive expression of theSASP-C gene from Bacillus megaterium and which, when present in multiplecopies, for example following infection of target cells, drives toxiclevels of SASP expression.

Bacteriophage vectors modified to contain a SASP gene have generallybeen named SASPject vectors. Once the SASP gene has been delivered to atarget bacterium, SASP is produced inside those bacteria where it bindsto bacterial DNA and changes the conformation of the DNA from B-liketowards A-like. Production of sufficient SASP inside target bacterialcells causes a drop in viability of affected cells.

Bacteriophage have been used as medicines for the treatment of bacterialinfections since the 1920s or 30s. Generally, bacteriophage are specificto their bacterial host. Some bacteriophage are temperate and othersnon-temperate. Temperate phage are able to infect the host cell andintegrate into the host cell genome becoming a prophage which isgenerally harmless to the host cell in this state. Non-temperate or“lytic” phage are only able to replicate in a lytic lifestyle by makingnew bacteriophage progeny and ending in lysis of the host cell andrelease of mature phage particles. For useful medicines, the challengeis to provide bacteriophage compositions which can be used to treatinfection from a variety of different bacteria in an effective way. Itis commonly thought that this is achieved using the most potentbacteriophage compositions available: those with a broadened host range,possibly as a mixture or “cocktail” of bacteriophage (Carlton, 1999;Kutateladze and Adamia, 2010). Cocktails of wild type phage have beenused to ensure sufficient spectrum of activity against clinical strainsof bacteria (Burrowes and Harper, 2012). Such cocktails can consist ofup to 20 different and unrelated phage (Abedon 2008). As an alternativeto the cocktail approach, E. coli bacteriophage K1-5 has been isolated.This is a naturally-occurring obligately lytic phage which carries morethan one host range determinant (HRD) allowing it to infect andreplicate on both K1 and K5 strains of E. coli (Scholl et al, 2001).These phage are considered to be extra potent.

There remains a need to provide improved bacteriophage for use intreating bacterial infections in medicine as well as inhibiting orpreventing bacterial cell growth in medical and non-medical situations.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a modifiedbacteriophage capable of infecting a plurality of different targetbacteria, which bacteriophage includes a toxin gene encoding a toxinprotein which is toxic to the target bacteria; wherein the bacteriophageis lytic; and wherein the bacteriophage expresses host range determinant(HRD) proteins which have a plurality of bacterial host specificities.The bacterial host specificity of the HRD is advantageously within thesame bacterial species.

It has surprisingly been found that a modified bacteriophage may beproduced which is capable of infecting a variety of different targetbacteria, and which is effective for use in medicine when engineered tocarry a gene for a toxic protein. The modified bacteriophage is lethalto bacterial cells, and, despite being lytic, and thus requiringcompletion of the phage lifecycle to be released from cells, can bemanufactured in a host cell through several rounds of infection andreplication.

In one aspect, the HRD proteins which have a plurality of bacterial hostspecificities are hybrid HRD proteins each comprising an amino acidsequence from a plurality of different bacteriophages. Because themodified bacteriophage expresses a hybrid HRD protein this confers anenhanced host range on the phage. Bacteriophages according to theinvention may be produced by genetic engineering, for example byselecting HRD from closely related phage. Having created such anextra-potent lytic phage, it can then be engineered to create a vectorincorporating the toxin gene, which vector is capable of beingmanufactured in a host cell and effective as an antibacterial agent invivo.

In another aspect, the HRD proteins expressed by the bacteriophagecomprise a plurality of different HRDs, wherein each HRD has a differentbacterial host specificity. In this aspect the HRDs can be homologous,heterologous, (hybrid) or a mixture of homologous and hybrid HRDs. Theplurality of different HRDs confers upon the bacteriophage an enhancedhost range. Such bacteriophage may be produced by genetic engineering,for example by selecting HRDs from phage which infect the same bacterialspecies. Having created such an extra-potent lytic phage, it can then beengineered to create a vector incorporating the toxin gene, which vectoris capable of being manufactured in a host cell an effective as anantibacterial agent in vivo.

In a preferred arrangement, the toxin gene comprises an α/β smallacid-soluble spore protein (SASP) gene encoding a SASP. In one aspect,the term ‘SASP’ as used in the specification refers to a protein withα/β-type SASP activity, that is, the ability to bind to DNA and modifyits structure from its B-like form towards A-like form, and not onlycovers the proteins listed in appendix 1 of WO02/40678, but also anyhomologues thereof, as well as any other protein also having α/β-typeSASP activity. In an alternative aspect, the term ‘SASP’ as used in thespecification refers to any protein listed in appendix 1 of WO02/40678,or any homologue having at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%,96%, 98% or 99% sequence identity with any one of the proteins. listedin appendix 1 of WO02/40678. In another alternative aspect, the term‘SASP’ as used in the specification refers to any protein listed inappendix 1 of WO02/40678.

The SASP gene may be chosen from any one of the genes encoding the SASPdisclosed in Appendix 1 of WO02/40678. In a preferred arrangement theSASP is SASP-C. The SASP-C may be from Bacillus megaterium.

It is preferred that the SASP gene is under the control of aconstitutive promoter which is advantageously sufficiently strong todrive production of toxic levels of SASP when the modified bacteriaphage is present in multiple copies in the target bacterium. Usefulconstitutive promoters include pdhA for pyruvate dehydrogenase E1component alpha sub units, rpsB for the 30S ribosomal protein S2, pgifor glucose-6-phosphate isomerase and the fructose bisphosphate aldolasegene promoter fda. Preferred regulated promoters, active duringinfection, are lasB for elastase. These promoters are typically from P.aeruginosa. Promoters having a sequence showing at least 90% sequenceidentity to these promoter sequences may also be used.

The present invention is generally applicable to bacteriophage infectinga variety of different target bacteria. In one arrangement at least oneof the target bacteria is Pseudomonas. Advantageously, the plurality ofdifferent target of bacteria is a plurality of different Pseudomonasbacteria. An important target is Pseudomonas aeruginosa.

It was previously considered that an obligate lytic phage would beunsuitable for use as a SASPject vector, since a requirement of aSASPject vector is that it is specifically not lytic for optimaltherapeutic use. A non-lytic vector allows an increased time window forSASP expression, thereby increasing the efficacy of the treatment. Inaddition, prevention of rapid lysis upon treatment in vivo, wasconsidered advantageous because it would limit the potential release ofantibiotic resistance genes and toxic cell wall components which canlead to a dangerous inflammatory response.

The approach described in the present invention is advantageous ascompared to the phage cocktail approach described previously. Mixturesof modified bacteriophage, such as SASPject vectors, are identical instructure and genome sequence, other than carrying a different HRD orhybrid HRD. One advantage is that control of the manufacturing processfor the mix of SASPjects will be straightforward, which is an importantaspect of a pharmaceutical preparation: the process will be materiallythe same for phage modified to carry a heterologous HRD as they shareidentical or near-identical biophysical properties. Another advantage isthat the in vivo characteristics of the SASPject vectors are likely tobe similar, e.g. pharmacokinetics/pharmacodynamics, as each vector isstructurally the same or similar.

In one aspect of the present invention it has been found that phage canbe created which are extra-potent obligately-lytic bacteriophagecarrying HRDs which have a plurality of bacterial host specificities.Surprisingly, such phage can be used to make enhanced SASPject vectorswhich retain a lytic phenotype, and remain effective in vivo, and suchphage can be manufactured to an adequate titre in a bacterial host.WO02/40678 and WO2009019293 have taught the creation of SASPject vectorsfrom bacteriophage which have been modified to remove one or more lysisgenes, and which reside as prophage in a host cell. Such SASPjectvectors are manufactured by prophage induction and the cells are lysedby the exogenous addition of lysis agents, e.g. cell wall degradingenzymes or chemicals such as chloroform. As such, the cells do not needto remain viable until the end of the phage lifecycle, as the finallysis step is not reliant upon the synthesis and accumulation in thehost cell of the modified phage's own lysis proteins. Given the toxicnature of the SASP protein, and the extra-potent nature of the modifiedphage described in the present invention, it was not anticipated thatsuch phage would be suitable for modification to create SASPjects whichcould be manufactured via an obligate lytic lifecycle, requiring thephage to complete its lytic lifecycle in order to create viable SASPjectvectors. However, it has been found that such SASPject vectors arecapable of replication in host cells to an adequate titre. This allowsthe manufacture of SASPject vectors in a quantity suitable for effectivein vivo use. Advantageously, SASPject vectors based upon such modifiedobligate lytic phage are capable of replication at the site ofinfection. This means that the dose required for a lytic SASPject can belower than that required for a non-lytic SASPject when used in vivo.Lytic SASPject vectors according to the invention may be considered morepotent than their non-lytic counterparts.

Phage suitable for such modification may be isolated by screening forphage capable of infecting a chosen bacterial species. For instance,phage may be isolated which infect Pseudomonas aeruginosa, by screeningenvironmental sources for phage which are able to form plaques onrepresentative P. aeruginosa strains (Gill and Hyman, 2010). Isolatedphage may have their whole genomes sequenced and annotated. HRDs may betail fibre proteins, which are commonly found to be proteins responsiblefor the initial recognition/binding to the host bacterium, for instancein phage T4, T5 and T7 (Rakhuba et al., 2010). Alternatively other HRDmay be baseplate proteins. Phage genomes may be searched for potentialHRD sequences by assessing the homology of all proteins in the phagegenome to known sequences, using BLAST searches.

According to the present invention it is preferred that each HRD has abroad host range. This may be defined as the ability to infect >50% of adiverse collection or clinical isolates, totaling at least 35,preferably at least 40, more preferably at least 44, and mostpreferably >50 in number. Such isolates should be from a range ofgeographical locations, including Europe, the Americas, and Asia, shouldcarry a diverse range of antibiotic resistance phenotypes, includingmulti-drug resistant (MDR) strains, and should be from a diverse rangeof infection sites, such as strains cultured from blood, lung and skininfections. Such isolates can be obtained from public strain collectionssuch as the American Type Culture Collection (ATCC) and the NationalCollection of Type Cultures (NCTC). Generally, each tail fibre proteincomprises a C-terminal receptor binding region for binding to the targetbacteria and an N-terminal region linking the C-terminal receptorbinding region to the body of the bacteriophage. Each of the C-terminaland N-terminal regions may be from different bacteriophage. In onearrangement, the N-terminal region comprises amino acids 1 to 628 of thetail fibre protein and the C-terminal region comprises the amino acids629 to 964 of the tail fibre protein.

The C-terminal region may have no more than 96% amino acid sequenceidentity with the C-terminal region of bacteriophage Phi33 and may befrom any one of the bacteriophage Phi33, LBL3, SPM-1, F8, PB1, KPP12,LMA2, SN, 14-1, JG024, NH-4, PTP47, C36, PTP92 and PTP93. Lower aminoacid sequence identities in the C-terminal region are preferred.Advantageously the sequence identity is less than 90%, moreadvantageously less than 80%, preferably less than 70%, more preferablyless than 60%, still more preferably less than 50%, particularlypreferably less than 40%, more particularly preferably less than 30%.The N-terminal region may have at least 90% and advantageously at least95% amino acid sequence identity with the N-terminal region ofbacteriophage Phi33 and may be from any one of bacteriophage Phi33,LBL3, SPM-1, F8, PB1, KPP12, LMA2, SN, 14-1, JG024, NH-4, PTP47, C36,PTP92 and PTP93. The N-terminal region and the C-terminal region may befrom the same bacteriophage to provide a homologous tail fibre protein.Alternatively, the N-terminal region and the C-terminal region may befrom different bacteriophage tail fibre proteins to provide aheterologous tail fibre protein. In one arrangement where the phage tailfibre protein is homologous, each tail fibre protein is from abacteriophage selected from Phi33, LBL3, SPM-1, F8, PB1, KPP12, LMA2,SN, 14-1, JG024, NH-4, PTP47, C36, PTP92 and PTP93.

It is advantageous to identify phage tail fibre proteins which sharesequence identity of greater than 90% in the N-terminal region. Forexample several phage—Phi33, PTP47, PTP92 and C36—with a broad hostrange for P. aeruginosa strains (all of these phage infect >60%, whenanalysed against 260 strains), have been isolated/identified and theirgenomes sequenced. Analysis of the genome sequences of Phi33, PTP47,PTP92 and C36 reveals that they contain genes encoding putative tailfibre proteins with a high level of sequence identity in the N-terminalregion (>95% amino acid sequence identity), following a 2 sequence BLASTalignment, compared to the Phi33 tail fibre amino acids 1-628 (aminoacid identity in parentheses): C36 (96%), PTP47 (98%), PTP92 (97%).BLAST searches have shown that these 4 phages are related to 10 otherdeposited phage genome sequences which, together, form the family ofPB1-like phage: PB1, SPM1, F8, LBL3, KPP12, LMA2, SN, JG024, NH-4, 14-1(Ceyssens et al., 2009). The homology of these putative tail fibreproteins was assessed. Following a 2 sequence BLAST alignment, comparedto the Phi33 tail fibre protein (amino acid identity in parentheses):LBL3 (96%), SPM-1 (95%), F8 (95%), PB1 (95%), KPP12 (94%), LMA2 (94%),SN (87%), 14-1 (86%), JG024 (83%), NH-4 (83%), C36 (96%), PTP47 (86%),PTP92 (83%). An alignment of all 14 of the aforementioned phage tailfibre proteins is shown in FIG. 13 .

Analysis of the annotated tail fibre protein sequences from these 14phages reveals that the N-terminal region of the proteins—equivalent toPhi33 tail fibre amino acids 1-628—show an even higher level of sequenceidentity at the amino acid level than the sequence identity of theseproteins over their entire length, in the range of 96-100% for all 14proteins. Following a 2 sequence BLAST alignment, compared to theN-terminal amino acids 1-628 of the Phi33 tail fibre protein (amino acididentity in parentheses): LBL3 (96%), SPM-1 (96%), F8 (96%), PB1 (96%),KPP12 (98%), LMA2 (99%), SN (99%), 14-1 (97%), JG024 (97%), NH-4 (97%),PTP47 (98%), C36 (96%), PTP92 (97%). However, the C-terminal region ofthe protein—equivalent to Phi33 tail fibre amino acids 629-964—is not asconserved as the N-terminal region in some of the proteins, the range ofsequence identity being typically 57-96%. Following a 2 sequence BLASTalignment, compared to the C-terminal 629-964 amino acids of the Phi33tail fibre protein (amino acid identity in parentheses): LBL3 (94%),SPM-1 (93%), F8 (93%), PB1 (94%), KPP12 (87%), LMA2 (85%), SN (65%),14-1 (65%), JG024 (57%), NH-4 (57%), PTP47 (64%), C36 (96%), PTP92(57%). Analysis of phage tail fibres from other, well characterised,phage has shown that they possess an N-terminal tail base plate bindingregion and a C-terminal receptor binding region (Veesler and Cambillau,2011). In experimental analysis of their bacterial strain host range,using plaque assay or growth inhibition tests, the phage Phi33, PTP47,PTP92 and C36 have overlapping but non-identical host range (Table 1).Taken together with the established evidence for the role of theC-terminal region of phage tail fibres being involved in bacterial hostreceptor binding, and the sequence variation in the C-terminal region ofthese 4 phage, and their similar but non-identical host range, it ispostulated that the C-terminal variation is associated with host rangein the phage assessed.

It is further provided, according to this invention, that the genes fora homologous tail fibre protein can be taken from one phage and added toanother, replacing the resident tail fibre, based upon their high levelof sequence identity in the N-terminal region. The N-terminal region isthought to be involved in the binding of the tail fibre to the phagetail (Veesler and Cambillau, 2011), allowing the formation of viablephage with the host range associated with donor phage's tail fibre.Hybrid tail fibre genes may be made, carrying the conserved N-terminaltail attachment region of the tail fibre from a recipient phage,together with the variable C-terminal receptor-binding region from ahomologous donor phage tail fibre protein, using tail fibres genes suchas those described herein. Such tail fibre hybrid genes could be used toreplace the tail fibres of the phage. This provides an N-terminal regionof the hybrid tail fibre (from the recipient phage) and allows theformation of viable phage with the host range associated with donorphage's tail fibre C-terminal receptor-binding region. Transplantationof engineered tail fibre hybrid genes into a recipient phage has beendemonstrated in the present invention. Using standard molecular genetictechniques, Phi33 has been modified to carry heterologous tail fibrehybrids from the following phage: PTP92, PTP47, LBL3, SPM-1, F8, PB1,KPP12, LMA2, SN, 14-1, NH-4. All modified phage have been shown to beviable and able to plaque on P. aeruginosa. (The nomenclature of tailfibre hybrids is as follows: As an example, a hybrid gene such that theN-terminal tail attachment region of Phi33 is hybridised with theC-terminal receptor binding region of PTP47 is Phi33(N)PTP47(C).)

In one such modified phage, Phi33 was engineered such that its tailfibre gene carries the C-terminal receptor binding region of PTP92,creating PTP93 (Phi33(N)PTP92(C)). This was assessed in more detail, bytesting the host range against 35 diverse P. aeruginosa clinicalisolates. Comparing host range of the progenitor phage (Phi33), the tailfibre donor (PTP92) and the hybrid phage (PTP93), the host range of thePTP93 hybrid phage is equivalent to that of the tail fibre donor phage(PTP92) rather than Phi33, but it was surprisingly found that in someinstances PTP93 possesses the host range of Phi33 on strains that PTP92cannot infect, thus PTP93 inherits the host range of both phages (Table2). PTP93 possesses a broader host range (92%) than either Phi33 (74%)or PTP92 (66%) (Table 2). PTP93 is an example of an obligately lyticbacteriophage which can be considered as “extra-potent” as it possessesa characteristic above and beyond those exhibited in their unmodifiedstate. Such extra potent phage are suitable for further modification tomake SASPject vectors.

Phi33, through assessment of sequence homology by one skilled in theart, can be placed in the group of PB1-like phage (Ceyssens et al.,2009), and may be considered a broad host range phage by the definitiongiven earlier. Such a phage could be isolated by the methods describedearlier. Phi33 is suitable for genetic modification to introduce a genefor SASP under the control of a promoter. Such a modified phage would besuitable for use in treating infections caused by susceptible P.aeruginosa bacteria, either on its own or in combination with othermodified bacteriophage. In one arrangement, the invention may not extendto an isolated bacteriophage PTPX31 or PTPX32.

A preferred approach according to the present invention is to use one ormore obligately lytic phage, each engineered to carry a SASP geneexpressed from a constitutive promoter, each phage being geneticallyidentical other than their tail fibre or tail fibre hybrid gene.Preferred obligately lytic phage for modification and for provision oftail fibre genes to create tail fibre genes or tail fibre hybrid genesare phages carrying tail fibre genes which encode predicted proteinsthat possess ≥90% amino acid sequence identity in their N-terminalregions compared to N-terminal regions of the tail fibre of otherisolated or identified phage.

Another preferred approach according to the present invention is to useone or more obligately lytic phage engineered to express 2 or more HRDs(extra potent obligately lytic phage), each engineered to carry a SASPgene expressed from a constitutive promoter, each phage beinggenetically identical other than carrying different tail fibre genes, ortail fibre hybrid genes, Preferred obligately lytic phages formodification and for provision of tail fibre genes to create phagescarrying multiple tail fibre genes or tail fibre hybrid genes are phagescarrying tail fibre genes which encode predicted proteins that possess≥90% amino acid sequence identity in their N-terminal regions comparedto N-terminal regions of the tail fibre of other isolated or identifiedphage.

Preferred obligate lytic phage meeting these criteria are Phi33, PTP92,PTP47, LBL3, SPM-1, F8, PB1, KPP12, LMA2, SN, 14-1, NH-4, PTP93, JG024,PTP47 and C36. Such phage can be identified by a simple PCR assay, bysubjecting plaques of isolated phage to PCR with primers specific tohighly conserved regions in the N-terminal region of the tail genes. Insuch a way, suitable phage can be identified without whole genomesequencing. Phage PB1 can be obtained from a public strain collection.Phages need not be isolated or provided in order to generate tail fibresequences as such sequences may be identified in DNA sequence databases,or other sources of DNA sequences, which may provide the informationnecessary in order to synthesise and clone, by standard methods, suchsequences, or to create hybrid tail fibre sequences.

Particularly preferred phage for modification are PTP93, Phi33, PTP92,PTP47 and C36. Particularly preferred extra-potent obligate lytic phageare: Phi33, modified to carry the tail fibre hybrid Phi33(N)PTP92(C) inplace of the resident tail fibre; Phi33 modified to carry the tail fibrehybrid Phi33(N)PTP47(C) in place of the resident tail fibre. In oneaspect of the present invention, preferred extra-potent non-lyticSASPject derivatives of Phi33 include: Phi33, modified to carry the tailfibre hybrid Phi33(N)PTP92(C) in place of the resident tail fibre andcarrying SASP-C from Bacillus megaterium, codon-optimised for expressionin P. aeruginosa, under the control of the P. aeruginosa fructosebisphosphate aldolase (fda) gene promoter; Phi33 modified to carry thetail fibre hybrid Phi33(N)PTP47(C) in place of the resident tail fibreand carrying SASP-C from Bacillus megaterium, codon-optimised forexpression in P. aeruginosa, under the control of the P. aeruginosafructose bisphosphate aldolase (fda) gene; Phi33 modified to carrySASP-C from Bacillus megaterium, codon-optimised for expression in P.aeruginosa, under the control of the P. aeruginosa fructose bisphosphatealdolase (fda) gene promoter. In a particularly preferred aspect thepresent invention provides a mixture of SASPject comprising orconsisting of the 3 aforementioned SASPjects formulated together.

A mixture of three modified bacteriophage, designated PT3.9, wasconstructed and its efficacy in killing P. aeruginosa tested. Themixture consists of: Phi33 carrying the Phi33(N)PTP92(C) tail fibre,modified to carry the fda-SASP-C (P. aeruginosa codon optimisedsequence) (PTP388); Phi33 carrying the Phi33(N)PTP47(C) tail fibre,modified to carry the fda-SASP-C (P. aeruginosa codon optimisedsequence) (PTP389); Phi33 carrying the Phi33(N)Phi33(C) tail fibre,modified to carry the fda-SASP-C (P. aeruginosa codon optimisedsequence) (PTP387).

The efficacy of PT3.9 was tested in a 6 hour time-kill curve experimentagainst a multi-drug resistant (MDR) clinical isolate (trachea isolationsite, antibiotic resistance to ceftazidime, piperacillin-tazobactam andimipenem) of P. aeruginosa, strain 3503 and reference strain ATCC 27853.Briefly, cultures were set up in Luria Bertani (LB) broth supplementedwith 5 mM calcium chloride, 5 mM magnesium sulphate and 0.1% glucose (LCbroth), and grown at 37° C. 5×10⁵ colony forming units per millilitre(cfu/ml) of P. aeruginosa was incubated with 3×10⁹ plaque forming unitsper ml (pfu/ml) of PT3.9, or extra LC broth as a control (untreatedculture). Samples were removed at 0, 1, 2, 4, and 6 hours for serialdilution and plating on LC agar plates and then overnight incubation at32° C. For both strains, the viable cell count was reduced from 5×10⁵cfu/ml to below the limit of detection (10² cfu/ml) within 1 hour oftreatment, and no viable cells were detected after 6 hours (FIG. 14 ).In contrast, the untreated control culture grew to between 5×10⁸ and1×10⁹ cfu/ml for both strains. This demonstrates the ability of PT3.9 tokill clinical strains of P. aeruginosa.

PTP387, a component SASPject of PT3.9, was tested in vivo in comparisonto PTP284, a SASPject which is equivalent to PTP387 other than lackingone of the lysis genes (endolysin). An immunocompetent murine model ofP. aeruginosa bacteraemia was used to assess the potency of the twoSASPject vectors. Briefly, animals were infected by intravenousinjection with an inoculum of 7.5 log₁₀ cfu of P. aeruginosa strain ATCC27853. At 2 hours post infection, the mice were treated either withPTP284, PTP387, vehicle (buffer), or ceftazidime (50 mg/kg) byintravenous injection. PTP284 and PTP387 were administered at thefollowing doses: 3×10¹⁰, 1×10¹⁰, 3×10⁹, and 1×10⁹ pfu. At 22 hours posttreatment, mice were euthanised by carbon dioxide asphyxiation, and thelivers were removed and weighed. Liver tissue was homogenised intryptone soya broth (TSB) and the number of viable cells in the livertissue was enumerated by serial dilution and plating on Cetrimide agarplates. A control (not treated) group of mice was euthanised at 2 hourspost infection and the viable cells in the liver tissue enumerated, toassess the viable cell count in the lung tissue at the time oftreatment. A dose response was seen for both PTP284 and PTP387, whenassessing the geometric mean of the bioburden levels of P. aeruginosa inliver tissue (FIG. 15 ). The potency of PTP387 was greater than that ofPTP284 when assessing the log reduction in cfu/g liver tissue comparedto the level assessed at the time of treatment: 22 hours post treatment,PTP387 caused a 3-log reduction in liver tissue bioburden at a 1×10⁹ pfudose, whilst an equivalent effect of PTP284 was seen at 1×10¹⁰ pfu.PTP387 caused a greater maximum effect on liver tissue bioburdenreduction compared to PTP284: at a dose of 3×10¹⁰ pfu, PTP387 caused a5.4-log reduction in liver tissue bioburden at 22 hours post treatment,whilst PTP284 caused a 2.4-log reduction. This demonstrates theincreased potency of the lytic SASPject in vivo when compared toequivalent non-lytic SASPject.

The mixture of lytic modified SASPject phage comprising PT3.9 was testedin vivo in comparison to the mixture of non-lytic modified SASPjectphage comprising PT3.8. The modified phage in PT3.8 are identical tothose in PT3.9 other than lacking the lysis gene (endolysin) present inPT3.9. A neutropenic murine model of P. aeruginosa pneumonia was used toassess the potency of the two mixtures of SASPject phage. Briefly,animals were rendered neutropenic by immunosuppression withcyclophosphamide 200 mg/kg 4 days before infection and 150 mg/kg 1 daybefore infection by intraperitoneal injection. Animals wereanaesthetised and infected intranasally with 4.5×10³ cfu of P.aeruginosa strain ATCC 27853. Groups of 6 mice were given eachtreatment. PT3.8 and PT3.9 were administered via the trachea 15 minutespost infection in an aerosolised form at the following doses: 1×10⁹,3×10⁹, 1×10¹⁰ and 1.5×10¹⁰ pfu. Tobramycin (200 μg) was administered inan aerosolised form to a control group of mice, and another group wastreated with placebo buffer. Animals were euthanised 24 hours posttreatment and lung tissue was removed and homogenised in phosphatebuffered saline (PBS). The tissue homogenate was serially diluted andplated on agar plates to enumerate the bacteria in the lung tissue. Acontrol (not treated) group of mice was euthanised at 15 minutes postinfection and the viable cells in the lung tissue enumerated, to assessthe viable cell count in the tissue at the time of treatment. A doseresponse was seen for both PT3.8 and PT3.9, when assessing the geometricmean of the bioburden levels of P. aeruginosa in lung tissue (FIG. 16 ).The potency of PT3.9 was greater than that of PT3.8. When assessing thelog reduction in cfu/g lung tissue compared to the level assessed at thetime of treatment: 24 hours post treatment, PT3.9 caused a >2-logreduction in lung tissue bioburden at a 1×10⁹ pfu dose, whilst anequivalent effect of PT3.8 was seen at doses between 1×10¹⁰ and 1.5×10¹⁰pfu. Thus PT3.9 was more than 10 times more potent than PT3.8.

The ability of PTP387 to propagate in a host cell and yield bacteriallysates suitable for further purification was assessed in comparison tothe unmodified phage (Phi33). Briefly, overnight culture of P.aeruginosa strain 1868 were used to inoculate 2×1 L bioreactor vessels,each containing 0.5 L of rich broth medium, to an OD600 of 0.05. Thecultures were grown to OD600=0.3-0.4 and then infected with phage Phi33or PTP387 to a final concentration of 1×10⁷ pfu/ml, and grown further at37° C. After 5 hours of growth, the cultures were treated with benzonaseand sterile filtered. The concentration of each phage was determined byplaque assay. The lysate titres yielded for PTP387 were comparable tothose obtained for Phi33: 2×10¹¹ pfu/ml (PTP387) and 5×10¹¹ pfu/ml(Phi33). The titre of PTP387 increased by 4-logs over 5 hours,demonstrating the ability of this phage to complete several cycles ofinfection, multiplication and lysis, despite the presence of the toxicSASP gene.

Fermentations were performed at 15.5-16 L scale for all 3 of themodified phage which comprise PT3.9. Briefly, overnight cultures ofPseudomonas aeruginosa were used to inoculate a 16 L bioreactorcontaining 15.5 to 16 L of rich broth medium, to an OD600 of 0.05. Eachculture was grown to OD600=0.4-0.7 before being infected with eitherPTP387, PTP388 or PTP389 (SASPject phage which comprise PT3.9) to afinal concentration of 1×10⁷ pfu/ml. Cultures were grown for another 5hours before Benonzase treatment. Samples were removed, sterilefiltered, and analysed by plaque assay. Two fermentations were performedfor each SASPject phage. All 3 of the modified SASPject phage comprisingPT3.9 were able to propagate to 5×10¹¹ to 1×10¹² pfu/ml. Thus all 3 ofthe modified SASPject phage are able to complete several cycles ofinfection, multiplication and lysis, despite the presence of the toxicSASP gene.

The quantities of PTP387 obtained by such manufacturing processes at 1 Lscale are suitable for effective in vivo use, as demonstrated by the useof such preparations in murine infection models (FIGS. 15 and 16 ). Thelevels of PT387, PTP388 and PTP389 obtained by such manufacturingprocesses at 15.5-16 L scale are suitable for effective in vivo use, asdemonstrated by the use of such preparations, when combined as a mixture(PT3.9), in murine infection models (FIGS. 15 and 16 ).

In another embodiment, an obligately lytic phage may be modified tocreate a SASPject by inserting a SASP gene under the control of aconstitutive promoter, and the tail fibre gene could be deletedaltogether. Such phage must be propagated in a strain in which a tailfibre gene or tail fibre hybrid gene is expressed in trans. In such aninstance, the SASPject progeny from such a strain would carry a singletail fibre, derived from the propagation strain, yet would lack in theirgenomes any tail fibre or tail fibre hybrid gene(s). Several suchpropagation strains could be constructed and the same tail fibre deletedSASPject expressed in each. In this way several different SASPjectderivatives could be made, each carrying a different tail fibre or tailfibre hybrid protein. These SASPjects could be used to formulate amixture.

In another embodiment, an obligately-lytic phage engineered to carry aSASP gene expressed from a constitutive promoter may be propagated in ahost strain carrying the gene(s) for hybrid tail fibre protein(s) intrans under the control of a suitable promoter. Suitable promoters forthe tail fibre hydrid gene(s) may be a phage promoter, particularly thepromoter which drives expression of the tail fibre gene in theengineered, obligately-lytic phage. Other suitable promoters areinducible promoters, such as lac, and trp, together with their cognateregulatory proteins. The SASPject progeny obtained from such strains areextra-potent, carrying the tail fibre hybrid(s) expressed from thestrain in trans as well as their own. Alternatively, the tail fibre genefrom the obligately lytic phage may be deleted altogether, providingthat a strain is used for propagation in which tail fibre gene(s) ortail fibre hybrid gene(s) are expressed in trans, allowing for theformation of derivative SASPjects. In such an instance, the SASPjectprogeny from such a strain would carry multiple tail fibres, yet wouldlack in their genomes any tail fibre or tail fibre hybrid gene(s).

In a further aspect, the present invention provides a composition forinhibiting or preventing bacterial cell growth, which comprises amodified bacteriophage or mixtures thereof as defined herein and acarrier therefor. The modified bacteriophage may be provided inadmixture with at least one other modified bacteriophage which iscapable of infecting target bacteria, which includes a toxin gene suchas a SASP gene encoding a SASP which is toxic to the target bacteria.Then at least one other modified bacteriophage may or may not express aplurality of different HRDs. Such compositions may have a wide range ofuses and are therefore to be formulated according to the intended use.The composition may be formulated as a medicament, especially for humantreatment and may treat various conditions, including bacterialinfections. Among those infections treatable according to the presentinvention are localised tissue and organ infections, or multi-organinfections, including blood-stream infections, topical infections, oralinfections including dental carries, respiratory infections and eyeinfections. The carrier may be a pharmaceutically-acceptable excipientor diluent. The exact nature and quantities of the components of suchcompositions may be determined empirically and will depend in part uponthe routes of administration of the composition.

Routes of administration to recipients include intravenous,intra-arterial, oral, buccal, sublingual, intranasal, by inhalation,topical (including ophthalmic), intra-muscular, subcutaneous,intra-vaginal, intrathecal and intra-articular. For convenience of use,dosages according to the invention will depend on the site and type ofinfection to be treated or prevented. Respiratory infections may betreated by inhalation administration and eye infections may be treatedusing eye drops. Oral hygiene products containing the modifiedbacteriophage are also provided; a mouthwash or toothpaste may be usedwhich contains modified bacteriophage according to the inventionformulated to eliminate bacteria associated with dental plaqueformation.

A modified bacteriophage, or mixture thereof, according to the inventionmay be used as a bacterial decontaminant, for example in the treatmentof surface bacterial contamination as well as land remediation or watertreatment. The bacteriophage may be used in the treatment of medicalpersonnel and/or patients as a decontaminating agent, for example in ahandwash. Treatment of work surfaces and equipment is also provided,especially that used in hospital procedures or in food preparation. Oneparticular embodiment comprises a composition formulated for topical usefor preventing, eliminating or reducing carriage of bacteria andcontamination from one individual to another. This is important to limitthe transmission of microbial infections, particularly in a hospitalenvironment where bacteria resistant to conventional antibiotics areprevalent. For such a use the modified bacteriophage may be contained inTris buffered saline or phosphate buffered saline or may be formulatedwithin a gel or cream. For multiple use a preservative may be added.Alternatively the product may be lyophilised and excipients, for examplea sugar such as sucrose, may be added.

DETAILED DESCRIPTION OF THE INVENTION

This invention will now be described in more detail, by way of exampleonly, and with reference to the accompanying drawings, in which:

FIGS. 1A and 1B are a schematic diagram showing construction of aplasmid containing lacZΔM15

FIGS. 2A, 2B, 2C, and 2D are a schematic diagram showing construction ofplasmids with replaced tail fibre sections;

FIGS. 3A, 3B, 3C, and 3D are a schematic diagram showing construction ofphage with hybrid tail fibre genes, which may be subsequently modifiedto introduce SASP-C according to the invention;

FIGS. 4A, 4B, 4C, and 4D are a schematic diagram showing construction ofphage with further hybrid tail fibre genes, which may be subsequentlymodified to introduce SASP-C according to the invention;

FIGS. 5A and 5B are a schematic diagram showing construction ofbacteriophage with hybrid tail fibre genes, in which the lacZα markerhas been removed;

FIGS. 6A, 6B, 6C, and 6D are a schematic diagram showing construction ofplasmids in which SASP-C is introduced into a suitable Phi33 insertionsite;

FIGS. 7A and 7B are a schematic diagram showing production of furtherbacteriophage according to the invention;

FIGS. 8A, 8B, 8C, and 8D are a schematic diagram showing construction ofplasmids in which SASP-C codon optimised for expression in P. aeruginosais introduced into a suitable Phi33 insertion site;

FIG. 9 shows the sequence of the SASP-C gene from Bacillus megateriumstrain KM (ATCC 13632), which has been codon optimised for expression inP. aeruginosa; and contains SEQ ID NO: 59;

FIGS. 10A, 10B, 10C, and 10D are a schematic diagram showing productionof bacteriophage in which SASP-C which has been codon optimised forexpression in P. aeruginosa is introduced into a suitable Phi33insertion site;

FIGS. 11A, 11B, 11C, and 11D are a schematic diagram showing productionof further bacteriophage according to the invention;

FIGS. 12A and 12B are a schematic diagram showing production of furtherbacteriophage according to the invention;

FIGS. 13A, 13B, 13C, 13D and 13E are a CLUSTAL 2.1 multiple sequencealignment of the tail fibre proteins from Phage SPM-1, F8, PB1, C36,LBL3, Phi33, LMA2, KPP12, JG024, PTP92, NH-4, 14-1, PTP47, SN; andcontains SEQ ID NOs: 45-58; and

FIGS. 14A-14B. 24 hour time-kill curve showing the in vitro efficacy ofPT3.9 against P. aeruginosa strains 3503 (FIG. 14A) and ATCC 27853 (FIG.14B). Cultures were grown in Luria bertani (LB) broth supplemented with10 mM calcium chloride, 1 mM magnesium sulphate and 1% glucose, at 37°C.

FIG. 15 . In vivo efficacy of PT3.9 in a murine bacteraemia model ofinfection. Mice were treated IV with vehicle (tris buffered salinecontaining 1 mM magnesium sulphate, 10 mM calcium chloride and 10% v/vglycerol), PTP284 or PTP387 (2 hours post infection with P. aeruginosa).The viable cell counts in liver tissue at 22 hours post treatment areshown for each animal in each group (group size=6), the geomean for eachdata set is represented by a horizontal line.

FIG. 16 . In vivo efficacy of PT3.9 in a neutropenic murine pneumoniamodel of infection. Mice were treated IT (intra-trachea) with placebovehicle (tris buffered saline containing 1 mM magnesium sulphate, 10 mMcalcium chloride and 10% v/v glycerol), PT3.9, PT3.8 or Tobramycin (15minutes post infection with P. aeruginosa). The viable cell counts inlung tissue at 24 hours post treatment are shown for each animal in eachgroup (group size=6), the geomean for each data set is represented by ahorizontal line.

The product of the invention provides in one aspect a single tail fibrewithin an individual phage, or a mix of phages where each type of phagehas a single, different tail fibre.

This is a summary of the genetic modification of a lytic bacteriophageto render it non-lytic, such that it carries one of a number of possibletail fibre variants, in addition to SASP-C under the control of apromoter that usually controls expression of the Pseudomonas aeruginosa30S ribosomal subunit protein S2 gene (rpsB), or SASP-C codon optimisedfor expression in P. aeruginosa, under the control of a promoter thatusually controls expression of the P. aeruginosafructose-1,6-bisphosphate aldolase gene (fda).

Genes can be removed and added to the phage genome using homologousrecombination. There are several ways in which phages carrying foreigngenes and promoters can be constructed and the following is an exampleof such methods.

For the construction of a Phi33 derivative it is shown how, using an E.coli/P. aeruginosa broad host range vector, as an example only, theexisting tail fibre, or a section of the tail fibre, in thebacteriophage genome may be replaced by an alternative tail fibre ortail fibre section from a different bacteriophage, via homologousrecombination. It is also shown as an example only, how additional DNAsequences, such as the SASP-C gene from B. megaterium under the controlof a P. aeruginosa rpsB promoter, or the SASP-C gene from B. megaterium,codon optimised for expression in P. aeruginosa, under the control of aP. aeruginosa fda promoter may be added to the bacteriophage genome viahomologous recombination.

A tail fibre gene, or section of a tail fibre gene, from an alternativephage may be cloned between two regions of Phi33 DNA that flank thenative tail fibre, or section thereof, along with a lacZα geneticmarker, in a broad host range E. coli/P. aeruginosa vector. This plasmidmay be introduced into P. aeruginosa, and the resulting strain infectedwith Phi33. Following harvesting of progeny phage, double recombinantsin which the native Phi33 tail fibre or tail fibre section, has beenreplaced by the new tail fibre or tail fibre section and lacZα, may beisolated by plaquing on a suitable P. aeruginosa (lacZΔM15+) host strainusing medium containing a chromogenic substrate that detects the actionof β-galactosidase.

In a subsequent step, the lacZα marker may be removed from thebacteriophage genomes by making versions of the previously describedtail fibre region recombination plasmids that do not contain the lacZαmarker, introducing the new plasmids into suitable P. aeruginosa hoststrains and infecting with the previously modified bacteriophagederivatives of Phi33 carrying the corresponding alternative tail fibregene, or section thereof, along with the lacZα marker. Recombinants thatretain the new tail fibre or tail fibre section, but from which lacZαhas been removed, may be isolated by plaquing on a suitable P.aeruginosa (lacZΔM15+) host strain using medium containing a chromogenicsubstrate that detects the action of β-galactosidase.

In a subsequent step, a similar homologous recombination may be used tointroduce the gene for SASP-C, under the control of a P. aeruginosa rpsBpromoter, while simultaneously adding an E. coli lacZα reporter gene forthe identification of recombinant phage, into Phi33, or any of thepreviously described Phi33 derivatives, or similar bacteriophage orsimilar derivatives. A region consisting of SASP-C controlled by therpsB promoter, and the E. coli lacZα allele, may be cloned between tworegions of Phi33 that flank a suitable insertion site, such as theintergenic region located immediately downstream of the Phi33 tail fibreoperon, in a broad host range E. coli/P. aeruginosa vector. This plasmidmay be transferred to a suitable P. aeruginosa (lacZΔM15+) strain, andthe resulting strain infected by Phi33 or the previously constructedPhi33 derivative (from which the initial lacZα marker has been removed).Progeny phage may be harvested and double recombinants identified byplaquing on P. aeruginosa (lacZΔM15+), looking for acquisition of thenew lacZα reporter on medium containing a chromogenic substrate thatdetects the action of β-galactosidase.

In an alternative subsequent step, a similar homologous recombinationmay be used to introduce the gene for SASP-C that has been codonoptimised for expression in P. aeruginosa, under the control of a P.aeruginosa fda promoter, while simultaneously adding an E. coli lacZαreporter gene for the identification of recombinant phage, into Phi33,or any of the previously described Phi33 derivatives, or similarbacteriophage or similar derivatives. A region consisting of codonoptimised SASP-C controlled by the fda promoter, and the E. coli lacZαallele, may be cloned between two regions of Phi33 that flank a suitableinsertion site, such as the intergenic region located immediatelydownstream of the Phi33 tail fibre operon, in a broad host range E.coli/P. aeruginosa vector. This plasmid may be transferred to a suitableP. aeruginosa (lacZΔM15+) strain, and the resulting strain infected byPhi33 or the previously constructed Phi33 derivative (from which theinitial lacZα marker has been removed). Progeny phage may be harvestedand double recombinants identified by plaquing on P. aeruginosa(lacZΔM15+), looking for acquisition of the new lacZα reporter on mediumcontaining a chromogenic substrate that detects the action ofβ-galactosidase.

Since these bacteriophage to be modified are lytic (rather thantemperate), another requirement for these described steps ofbacteriophage construction is the construction of a suitable host P.aeruginosa strain that carries the E. coli lacZΔM15 gene at a suitablelocation in the bacterial genome, to complement the lacZα phenotypes ofthe desired recombinant bacteriophage. As an example, the constructionof these P. aeruginosa strains may be achieved via homologousrecombination using an E. coli vector that is unable to replicate in P.aeruginosa. The genomic location for insertion of the lacZΔM15 transgeneshould be chosen such that no essential genes are affected and nounwanted phenotypes are generated through polar effects on theexpression of adjacent genes. As an example, one such location may beimmediately downstream of the P. aeruginosa strain PAO1 phoA homologue.

The E. coli lacZΔM15 allele may be cloned into an E. coli vector that isunable to replicate in P. aeruginosa, between two regions of P.aeruginosa strain PAO1 genomic DNA that flank the 3′ end of phoA. Thisplasmid may be introduced into P. aeruginosa and isolates havingundergone a single homologous recombination to integrate the wholeplasmid into the genome selected according to the acquisition oftetracycline (50 μg/ml) resistance. Isolates (lacZΔM15+) which haveundergone a second homologous recombination event may then be isolatedon medium containing 10% sucrose (utilising the sacB counter-selectablemarker present on the plasmid backbone).

In a subsequent step, a similar homologous recombination may be used toremove the lacZα marker from the previously described, (lacZα+) Phi33derivatives that have been modified to introduce the gene for SASP-C,under the control of a P. aeruginosa rpsB promoter. A region consistingof SASP-C controlled by the rpsB promoter, may be cloned between tworegions of Phi33 that flank the chosen insertion site, in a broad hostrange E. coli/P. aeruginosa vector. This plasmid may be transferred to asuitable P. aeruginosa (lacZΔM15+) strain, and the resulting straininfected by the previously described (lacZα+)Phi33 derivatives that havebeen modified to introduce the gene for SASP-C, under the control of aP. aeruginosa rpsB promoter. Progeny phage may be harvested and doublerecombinants identified by plaquing on P. aeruginosa (lacZΔM15+),looking for loss of the lacZα reporter on medium containing achromogenic substrate that detects the action of β-galactosidase.

In an alternative subsequent step, a similar homologous recombinationmay be used to remove the lacZα marker from the previously described,(lacZα+) Phi33 derivatives that have been modified to introduce the genefor SASP-C, codon optimised for expression in P. aeruginosa, under thecontrol of a P. aeruginosa fda promoter. A region consisting of SASP-C,codon optimised for expression in P. aeruginosa, controlled by the fdapromoter, may be cloned between two regions of Phi33 that flank thechosen insertion site, in a broad host range E. coli/P. aeruginosavector. This plasmid may be transferred to a suitable P. aeruginosa(lacZΔM15+) strain, and the resulting strain infected by the previouslydescribed (lacZα+) Phi33 derivatives that have been modified tointroduce the gene for SASP-C, codon optimised for expression in P.aeruginosa, under the control of a P. aeruginosa fda promoter. Progenyphage may be harvested and double recombinants identified by plaquing onP. aeruginosa (lacZΔM15+), looking for loss of the lacZα reporter onmedium containing a chromogenic substrate that detects the action ofβ-galactosidase.

Experimental Procedures

PCR reactions to generate DNA for cloning purposes may be carried outusing Herculase II Fusion DNA polymerase (Agilent Technologies),depending upon the melting temperatures (Tm) of the primers, accordingto manufacturers instructions. PCR reactions for screening purposes maybe carried out using Taq DNA polymerase (NEB), depending upon the Tm ofthe primers, according to manufacturers instructions. Unless otherwisestated, general molecular biology techniques, such as restriction enzymedigestion, agarose gel electrophoresis, T4 DNA ligase-dependentligations, competent cell preparation and transformation may be basedupon methods described in Sambrook et al., (1989). Enzymes may bepurchased from New England Biolabs or Thermo Scientific. DNA may bepurified from enzyme reactions and prepared from cells using Qiagen DNApurification kits. Plasmids may be transferred from E. coli strains toP. aeruginosa strains by conjugation, mediated by the conjugation helperstrain E. coli HB101 (pRK2013). A chromogenic substrate forβ-galactosidase, S-gal, that upon digestion by β-galactosidase forms ablack precipitate when chelated with ferric iron, may be purchased fromSigma (S9811).

Primers may be obtained from Sigma Life Science. Where primers includerecognition sequences for restriction enzymes, additional 2-6nucleotides may be added at the 5′ end to ensure digestion of thePCR-amplified DNA.

All clonings, unless otherwise stated, may be achieved by ligating DNAsovernight with T4 DNA ligase and then transforming them into E. colicloning strains, such as DH5α or TOP10, with isolation on selectivemedium, as described elsewhere (Sambrook et al., 1989).

An E. coli/P. aeruginosa broad host range vector, such as pSM1080A, maybe used to transfer genes between E. coli and P. aeruginosa. pSM1080Awas previously produced by combining the broad host-range origin ofreplication from a P. aeruginosa plasmid, oriT from pRK2, the tetARselectable marker for use in both E. coli and P. aeruginosa, fromplasmid pRK415, and the high-copy-number, E. coli origin of replication,oriV, from plasmid pUC19.

An E. coli vector that is unable to replicate in P. aeruginosa, pSM1104,may be used to generate P. aeruginosa mutants by allelic exchange.pSM1104 was previously produced by combining oriT from pRK2, the tetARselectable marker for use in both E. coli and P. aeruginosa, fromplasmid pRK415, the high-copy-number, E. coli origin of replication,oriV, from plasmid pUC19, and the sacB gene from Bacillus subtilisstrain 168, under the control of a strong promoter, for use as acounter-selectable marker.

Detection of Phi33-Like Phage (PB1-Like Phage Family) ConservedN-Terminal Tail Fibre Regions by PCR

1. Primers for the detection of Phi33-like phage-like tail fibre genesin experimental phage samples may be designed as follows:

The DNA sequences of the tail fibre genes from all sequenced Phi33-likephage (including Phi33, PB1, NH-4, 14-1, LMA2, KPP12, JG024, F8, SPM-1,LBL3, PTP47, C36, PTP92 and SN) may be aligned using Clustal Omega,which is available on the EBI website, and the approximately 2 kb-longhighly conserved region mapping to the gene's 5′ sequence may be thusidentified (positions 31680-33557 in the PB1 genome sequence, Acc.EU716414). Sections of 100% identity among the 11 tail fibre genesequences may be identified by visual inspection. Three pairs of PCRprimers targeting selected absolutely conserved regions, and amplifyingPCR products no longer than 1 kb may be chosen as follows: pair B4500and B4501, defining a 193 bp-long region; pair B4502 and B4503, defininga 774 bp-long region; and pair B4504 and B4505, defining a 365 bp-longregion.

Primer B4500 consists of sequence of PB1 phage genome (Acc. EU716414)ranging from position 31680 to 31697. Primer B4501 consists of sequenceof PB1 phage genome (Acc. EU716414) ranging from position 31851 to31872. Primer B4502 consists of sequence of PB1 phage genome (Acc.EU716414) ranging from position 31785 to 31804. Primer B4503 consists ofsequence of PB1 phage genome (Acc. EU716414) ranging from position 32541to 32558. Primer B4504 consists of sequence of PB1 phage genome (Acc.EU716414) ranging from position 32868 to 32888. Primer B4505 consists ofsequence of PB1 phage genome (Acc. EU716414) ranging from position 33213to 33232.

B4500 (SEQ ID NO: 1) 5′-GTGATCACACCCGAACTG-3′ B4501 (SEQ ID NO: 2)5′-CGATGAAGAAGAGTTGGTTTTG-3′ B4502 (SEQ ID NO: 3)5′-ACGCCGGACTACGAAATCAG-3′ B4503 (SEQ ID NO: 4) 5′-TCCGGAGACGTTGATGGT-3′B4504 (SEQ ID NO: 5) 5′-CCTTTCATCGATTTCCACTTC-3′ B4505 (SEQ ID NO: 6)5′-TTCGTGGACGCCCAGTCCCA-3′

2. Phi33-like tail fibre genes may be detected in experimental phagesamples as follows:

Plaques of isolated phage of environmental origin may be picked fromagar plates and added to water and incubated for 30 minutes, makingplaque soak outs. The plaque soak outs may be diluted and a portionadded to PCR reactions containing one or all of the above primer pairs,and PCR may be performed according to a standard protocol. PCR productsmay be visualised on a 1.5% agarose gel with ethidium bromide staining,and evaluated for their size. PCR products of the correct size for theprimer pair used may be gel-extracted and submitted to an externalfacility for sequencing. Sequencing results may be compared with theavailable tail fibre gene sequences in order to confirm the identity ofthe PCR product.

Construction of Plasmids to Generate Pseudomonas aeruginosa StrainsCarrying the Escherichia coli lacZΔM15 Gene, Immediately Downstream ofthe phoA Locus of the Bacterial Genome

1. Plasmid pSMX200 (FIG. 1 ), comprising pSM1104 carrying DNA flankingthe 3′ end of the P. aeruginosa PAO1 phoA homologue, may be constructedas follows.

A region comprising the terminal approximately 1 kb of the phoA genefrom P. aeruginosa may be amplified by PCR using primers B4200 and B4201(FIG. 1 ). The PCR product may then be cleaned and digested with SpeIand BglII. A second region comprising approximately 1 kb downstream ofthe phoA gene from P. aeruginosa, including the 3′ end of the PA3297open reading frame, may be amplified by PCR using primers B4202 andB4203 (FIG. 1 ). This second PCR product may then be cleaned anddigested with BglII and XhoI. The two digests may be cleaned again andligated to pSM1104 that has been digested with SpeI and XhoI, in a 3-wayligation, to yield plasmid pSMX200 (FIG. 1 ).

Primer B4200 consists of a 5′ SpeI restriction site (underlined),followed by sequence located approximately 1 kb upstream of the stopcodon of phoA from P. aeruginosa strain PAO1 (FIG. 1 ). Primer B4201consists of 5′ BglII and AflII restriction sites (underlined), followedby sequence complementary to the end of the phoA gene from P. aeruginosastrain PA01 (the stop codon is in lower case; FIG. 1 ). Primer B4202consists of 5′ BglII and NheI restriction sites (underlined), followedby sequence immediately downstream of the stop codon of the phoA genefrom P. aeruginosa strain PAO1 (FIG. 1 ). Primer B4203 consists of a 5′XhoI restriction site (underlined), followed by sequence within thePA3297 open reading frame, approximately 1 kb downstream of the phoAgene from P. aeruginosa strain PAO1 (FIG. 1 ).

Primer B4200 (SEQ ID NO: 7) 5′-gataACTAGTCCTGGTCCACCGGGGTCAAG-3′Primer B4201 (SEQ ID NO: 8) 5′-gctcagatcttccttaagtcaGTCGCGCAGGTTCAG-3′Primer B4202 (SEQ ID NO: 9) 5′-aggaagatctgagctagcTCGGACCAGAACGAAAAAG-3′Primer B4203 (SEQ ID NO: 10) 5′-gataCTCGAGGCGGATGAACATTGAGGTG-3′

2. Plasmid pSMX203 (FIG. 1 ), comprising pSMX200 carrying lacZΔM15 underthe control of a lac promoter, may be constructed as follows.

The lacZΔM15 gene under the control of a lac promoter may be amplifiedby PCR from Escherichia coli strain DH10B using primers B4208 and B4209(FIG. 1 ). The resulting PCR product may then be digested with BglII andNheI, and ligated to pSMX200 that has also been digested with BglII andNheI, to yield plasmid pSMX203 (FIG. 1 ).

Primer B4208 consists of a 5′ BglII restriction site (underlined),followed by sequence of the lac promoter (FIG. 1 ). Primer B4209consists of a 5′ NheI restriction site (underlined), followed by abi-directional transcriptional terminator and sequence complementary tothe 3′ end of lacZΔM15 (underlined, in bold; FIG. 1 ).

Primer B4208 (SEQ ID NO: 11) 5′-gataagatctgagcgcaacgcaattaatgtg-3′Primer B4209 (SEQ ID NO: 12)5′-gatagctagcAGTCAAAAGCCTCCGGTCGGAGGCTTTTGACTTTATT TTTGACACCAGACCAAC-3′

Genetic modification of Pseudomonas aeruginosa to introduce theEscherichia coli lacZΔM15 gene immediately downstream of the phoA locusof the bacterial genome

-   -   1. Plasmid pSMX203 (FIG. 1 ) may be transferred to P. aeruginosa        by conjugation, selecting for primary recombinants by        acquisition of resistance to tetracycline (50 μg/ml).    -   2. Double recombinants may then be selected via sacB-mediated        counterselection, by plating onto medium containing 10% sucrose.    -   3. Isolates growing on 10% sucrose may then be screened by PCR        to confirm that lacZΔM15 has been introduced downstream of        the P. aeruginosa phoA gene.    -   4. Following verification of an isolate (PAX21), this strain may        then be used as a host for further modification of        bacteriophage, where complementation of a lacZα reporter is        required.        Construction of a Plasmid to Replace the 3′ Section of the Phi33        Tail Fibre with that of PTP92, Utilising a lacZα Screening        Process

1. pSMX284 (FIG. 2 ), comprising pSM1080A carrying the regionimmediately downstream of the Phi33 tail fibre gene, may be constructedas follows.

A 1 kb region of Phi33 sequence covering the terminal 20 bases of thePhi33 tail fibre, and the adjacent downstream region, may be amplifiedby PCR using primers B4222 and B4249 (FIG. 2 ). The resulting PCRproduct may then be cleaned and digested with NheI, and ligated topSM1080A that has also been digested with NheI and then treated withalkaline phosphatase prior to ligation, yielding plasmid pSMX284 (FIG. 2).

Primer B4222 consists of a 5′ NheI restriction site (underlined),followed by sequence from Phi33, approximately 1 kb downstream of theend of the Phi33 tail fibre gene (FIG. 2 ). B4249 consists of 5′NheI-KpnI-AvrII restriction sites (underlined), followed by sequencecomplementary to the 3′ end of the Phi33 tail fibre and sequenceimmediately downstream of the tail fibre open reading frame (FIG. 2 ).

B4222 (SEQ ID NO: 13) 5′-gataGCTAGCATGGTTTTCACGACCATG-3′ B4249(SEQ ID NO: 14) 5′-GATAGCTAGCGAGGTACCGACCTAGGTTTTCCAGCGAGTGACGTAAAATG-3′

2. pSMX285 (FIG. 2 ), comprising pSMX284 carrying lacZα, a 3′ section ofthe PTP92 tail fibre gene sequence, and a region of Phi33 sequencecomprising the 5′ end of the tail fibre gene and sequence locatedimmediately upstream of the Phi33 tail fibre gene, may be constructed asfollows.

The lacZα open reading frame may be amplified by PCR from pUC19 usingprimers B4250 and B4252 (FIG. 2 ). The PTP92 tail fibre 3′ section maybe amplified by PCR from PTP92 using primers B4251 and B4254 (FIG. 2 ).The lacZα open reading frame may then be joined to the PTP92 tail fibregene 3′ section by SOEing PCR using the outer primers, B4250 and B4254.A region comprising sequence of the 5′ end of the Phi33 tail fibre gene,and sequence located immediately upstream of the Phi33 tail fibre gene,may be amplified by PCR using primers B4253 and B4229 (FIG. 2 ). ThisPCR product may then be joined to the PCR product comprising lacZα andthe PTP92 tail fibre gene 3′ section, by SOEing PCR using the outerprimers B4250 and B4229. The resulting PCR product may then be cleanedand digested with AvrII and KpnI, and ligated to pSMX284 that has alsobeen digested with AvrII and KpnI, yielding plasmid pSMX285 (FIG. 2 ).

Primer B4250 consists of a 5′ AvrII restriction site, followed bysequence complementary to the 3′ end of the lacZα open reading frame(FIG. 2 ). Primer B4252 consists of a 5′ section of sequence thatoverlaps the 3′ end of the PTP92 tail fibre gene (underlined), followedby sequence of the 5′ end of the lacZα open reading frame. Primer B4251is the reverse complement of primer B4252 (FIG. 2 ). Primer B4254consists of 5′ sequence from within the Phi33 tail fibre gene(underlined), followed by sequence within the 3′ end of the PTP92 tailfibre gene (FIG. 2 ). Primer B4253 is the reverse complement of PrimerB4254. Primer B4229 consists of a 5′ KpnI restriction site (underlined),followed by sequence that is complementary to a region approximately 1kb upstream of the tail fibre gene in Phi33 (FIG. 2 ).

Primer B4250 (SEQ ID NO: 15) 5′-GataCCTAGGttagcgccattcgccattc-3′Primer B4252 (SEQ ID NO: 16)5′-CTATTCCAGCGGGTAACGTAAAatgaccatgattacggattC-3′ Primer B4251(SEQ ID NO: 17) 5′-GaatccgtaatcatggtcatTTTACGTTACCCGCTGGAATAG-3′Primer B4254 (SEQ ID NO: 18)5′-CAAGCGGGCCGGCTGGTCTCTCGGCAATAACTCCTATGTGATC-3′ Primer B4253(SEQ ID NO: 19) 5′-GATCACATAGGAGTTATTGCCGAGAGACCAGCCGGCCCGCTTG-3′Primer B4229 (SEQ ID NO: 20) 5′-gataGGTACCGCGACCGGTCTGTACTTC-3′

Genetic modification of Phi33 to replace the 3′ section of the tailfibre gene with that of PTP92

-   -   1. Plasmid pSMX285 (FIG. 2 ; FIG. 3 ) may be introduced into P.        aeruginosa strain PAX21 by conjugation, selecting        transconjugants on the basis of tetracycline resistance (50        μg/ml), yielding strain PTA80.    -   2. Strain PTA80 may be infected with phage Phi33, and the        progeny phage harvested.    -   3. Recombinant phage in which the 3′ end of the Phi33 tail fibre        gene has been replaced by that of PTP92, and to which lacZα has        been added, may be identified by plaquing the lysate from        step (2) on P. aeruginosa strain PAX21, onto medium containing        S-gal, looking for black plaques, which are indicative of        β-galactosidase activity.    -   4. PCR may be carried out to check that the tail fibre gene has        been replaced, and that lacZα is present.    -   5. Following identification of a verified isolate (PTPX81; FIG.        3 ), this isolate may be plaque purified twice more on P.        aeruginosa strain PAX21, prior to further use.        Construction of a Plasmid to Replace the 3′ Section of the Phi33        Tail Fibre with that of PTP47, Utilising a lacZα Screening        Process

1. pSMX286 (FIG. 2 ), comprising pSMX284 carrying lacZα, a 3′ section ofthe PTP47 tail fibre gene sequence, and a region of Phi33 sequencecomprising the 5′ end of the tail fibre gene and sequence locatedimmediately upstream of the Phi33 tail fibre gene, may be constructed asfollows.

The lacZα open reading frame may be amplified by PCR from pUC19 usingprimers B4250 and B4258 (FIG. 2 ). The PTP47 tail fibre 3′ section maybe amplified by PCR from PTP47 using primers B4259 and B4260 (FIG. 2 ).The lacZα open reading frame may then be joined to the PTP47 tail fibregene 3′ section by SOEing PCR using the outer primers, B4250 and B4260.A region comprising sequence of the 5′ end of the Phi33 tail fibre gene,and sequence located immediately upstream of the Phi33 tail fibre gene,may be amplified by PCR using primers B4261 and B4229 (FIG. 2 ). ThisPCR product may then be joined to the PCR product comprising lacZα andthe PTP47 tail fibre gene 3′ section, by SOEing PCR using the outerprimers B4250 and B4229. The resulting PCR product may then be cleanedand digested with AvrII and KpnI, and ligated to pSMX284 that has alsobeen digested with AvrII and KpnI, yielding plasmid pSMX286 (FIG. 2 ).

Primer B4250 consists of a 5′ AvrII restriction site, followed bysequence complementary to the 3′ end of the lacZα open reading frame(FIG. 2 ). Primer B4258 consists of a 5′ section of sequence thatoverlaps the 3′ end of the PTP47 tail fibre gene (underlined), followedby sequence of the 5′ end of the lacZα open reading frame. Primer B4259is the reverse complement of primer B4258 (FIG. 2 ). Primer B4260consists of 5′ sequence from within the Phi33 tail fibre gene(underlined), followed by sequence within the 3′ end of the PTP47 tailfibre gene (FIG. 2 ). Primer B4261 is the reverse complement of PrimerB4260. Primer B4229 consists of a 5′ KpnI restriction site (underlined),followed by sequence that is complementary to a region approximately 1kb upstream of the tail fibre gene in Phi33 (FIG. 2 ).

Primer B4250 (SEQ ID NO: 15) 5′-GataCCTAGGttagcgccattcgccattc-3′Primer B4258 (SEQ ID NO: 21)5′-CTTTTCCAGCGAGTGACGTAAAatgaccatgattacggattC-3′ Primer B4259(SEQ ID NO: 22) 5′-gaatccgtaatcatggtcatTTTACGTCACTCGCTGGAAAAG-3′Primer B4260 (SEQ ID NO: 23)5′-CAAGCGGGCCGGCTGGTCTCTCGGCAATAACTCCTATGTGATC-3′ Primer B4261(SEQ ID NO: 24) 5′-GATCACATAGGAGTTATTGCCGAGAGACCAGCCGGCCCGCTTG-3′Primer B4229 (SEQ ID NO: 20) 5′-gataGGTACCGCGACCGGTCTGTACTTC-3′Genetic Modification of Phi33 to Replace the 3′ Section of the TailFibre Gene with that of PTP47

-   -   1. Plasmid pSMX286 (FIG. 2 ; FIG. 4 ) may be introduced into P.        aeruginosa strain PAX21 by conjugation, selecting        transconjugants on the basis of tetracycline resistance (50        μg/ml), yielding strain PTA81.    -   2. Strain PTA81 may be infected with phage Phi33, and the        progeny phage harvested.    -   3. Recombinant phage in which the 3′ end of the Phi33 tail fibre        gene has been replaced by that of PTP47, and to which lacZα has        been added, may be identified by plaquing the lysate from        step (2) on P. aeruginosa strain PAX21, onto medium containing        S-gal, looking for black plaques, which are indicative of        β-galactosidase activity.    -   4. PCR may be carried out to check that the tail fibre gene has        been replaced, and that lacZα is present.    -   5. Following identification of a verified isolate (PTPX82; FIG.        4 ), this isolate may be plaque purified twice more on P.        aeruginosa strain PAX21, prior to further use.        Construction of a Plasmid to Remove the lacZα Marker from PTPX81

1. pSMX287 (FIG. 5 ), comprising pSM1080A carrying a 3′ section of thePTP92 tail fibre gene, and a region of Phi33 sequence locatedimmediately downstream of the Phi33 tail fibre gene, may be constructedas follows.

The region of Phi33 sequence located immediately downstream of the Phi33tail fibre may be amplified by PCR using primers B4222 and B4255 (FIG. 5). The 3′ end of the PTP92 tail fibre gene may be amplified by PCR usingprimers B4256 and B4257 (FIG. 5 ). These two PCR products may then bejoined by SOEing PCR, using the two outer primers B4222 and B4257. Theresulting PCR product may then be cleaned, digested with NheI, cleanedagain, and ligated to pSM1080A that has also been digested with NheI andthen treated with alkaline phosphatase prior to ligation, to yieldplasmid pSMX287 (FIG. 5 ).

Primer B4255 consists of a 5′ section of the end of the PTP92 tail fibregene (underlined), followed by sequence immediately downstream of thePhi33 tail fibre gene (FIG. 5 ). Primer B4256 is the reverse complementof primer B4255 (FIG. 5 ). Primer B4257 consists of a 5′ NheIrestriction site (underlined), followed by sequence of the terminal 1 kbof the PTP92 tail fibre gene (FIG. 5 ).

Primer B4255 (SEQ ID NO: 25)5′-CTATTCCAGCGGGTAACGTAAAATGAAATGGACGCGGATCAG-3′ Primer B4256(SEQ ID NO: 26) 5′-CTGATCCGCGTCCATTTCATTTTACGTTACCCGCTGGAATAG-3′Primers B4257 (SEQ ID NO: 27) 5′-gataGCTAGCGGCAATAACTCCTATGTGATC-3′Genetic Modification of PTPX81 to Remove the lacZα Marker

-   -   1. Plasmid pSMX287 (FIG. 5 ; FIG. 3 ) may be introduced into P.        aeruginosa strain PAX21 by conjugation, selecting        transconjugants on the basis of tetracycline resistance (50        μg/ml), yielding strain PTA82.    -   2. Strain PTA82 may be infected with phage PTPX81, and the        progeny phage harvested.    -   3. Recombinant phage in which the lacZα marker has been removed        may be identified by plaquing the lysate from step (2) on P.        aeruginosa strain PAX21, onto medium containing S-gal, looking        for white plaques, which are indicative of loss of        β-galactosidase activity.    -   4. PCR may be carried out to check that the tail fibre gene has        been retained, and that lacZα has been removed.    -   5. Following identification of a verified isolate (PTPX83; FIG.        3 ), this isolate may be plaque purified twice more on P.        aeruginosa strain PAX21, prior to further use.        Construction of a Plasmid to Remove the lacZα Marker from PTPX82

1. pSMX288 (FIG. 5 ), comprising pSM1080A carrying a 3′ section of thePTP47 tail fibre gene, and a region of Phi33 sequence locatedimmediately downstream of the Phi33 tail fibre gene, may be constructedas follows.

The region of Phi33 sequence located immediately downstream of the Phi33tail fibre may be amplified by PCR using primers B4222 and B4262 (FIG. 5). The 3′ end of the PTP47 tail fibre gene may be amplified by PCR usingprimers B4263 and B4264 (FIG. 5 ). These two PCR products may then bejoined by SOEing PCR, using the two outer primers B4222 and B4264. Theresulting PCR product may then be cleaned, digested with NheI, cleanedagain, and ligated to pSM1080A that has also been digested with NheI andthen treated with alkaline phosphatase prior to ligation, to yieldplasmid pSMX288 (FIG. 5 ).

Primer B4262 consists of a 5′ section of the end of the PTP47 tail fibregene (underlined), followed by sequence immediately downstream of thePhi33 tail fibre gene (FIG. 5 ). Primer B4263 is the reverse complementof primer B4262 (FIG. 5 ). Primer B4264 consists of a 5′ NheIrestriction site (underlined), followed by sequence of the terminal 1 kbof the PTP47 tail fibre gene (FIG. 5 ).

Primer B4262 (SEQ ID NO: 28)5′-CTTTTCCAGCGAGTGACGTAAAATGAAATGGACGCGGATCAG-3′ Primer B4263(SEQ ID NO: 29) 5′-CTGATCCGCGTCCATTTCATTTTACGTCACTCGCTGGAAAAG -3′Primers B4264 (SEQ ID NO: 30) 5′-gataGCTAGCGGCAATAACTCCTATGTGATC-3′Genetic Modification of PTPX82 to Remove the lacZα Marker

-   -   1. Plasmid pSMX288 (FIG. 5 ; FIG. 4 ) may be introduced into P.        aeruginosa strain PAX21 by conjugation, selecting        transconjugants on the basis of tetracycline resistance (50        μg/ml), yielding strain PTA83.    -   2. Strain PTA83 may be infected with phage PTPX82, and the        progeny phage harvested.    -   3. Recombinant phage in which the lacZα marker has been removed        may be identified by plaquing the lysate from step (2) on P.        aeruginosa strain PAX21, onto medium containing S-gal, looking        for white plaques, which are indicative of loss of        β-galactosidase activity.    -   4. PCR may be carried out to check that the tail fibre gene has        been retained, and that lacZα has been removed.    -   5. Following identification of a verified isolate (PTPX84; FIG.        4 ), this isolate may be plaque purified twice more on P.        aeruginosa strain PAX21, prior to further use.

Construction of a plasmid to introduce rpsB-SASP-C and lacZα into thephage genome of Phi33, PTPX83, PTPX84, and similar phage

1. Plasmid pSMX251 (FIG. 6 ), comprising pSM1080A containing regions ofPhi33 flanking the chosen insertion site for rpsB-SASP-C, such as theintergenic region immediately downstream of the tail fibre operon, maybe constructed as follows.

The region of Phi33 sequence immediately downstream of the choseninsertion site may be amplified by PCR using primers B4900 and B4901(FIG. 6 ). This PCR product may then be cleaned and digested with NheIand AvrII. The region of Phi33 sequence immediately upstream of thechosen insertion site may be amplified by PCR using primers B4902 andB4903 (FIG. 6 ). This second PCR product may then be cleaned anddigested with AvrII and NheI. The two PCR product digests may then becleaned again and ligated to pSM1080A that has been digested with NheIand treated with alkaline phosphatase prior to ligation. Clones carryingone insert of each of the two PCR products may be identified by PCRusing primers B4900 and B4903, and NheI restriction digest analysis ofthe purified putative clones, to identify plasmid pSMX251 (FIG. 6 ).

Primer B4900 consists of a 5′ NheI restriction site (underlined),followed by Phi33 sequence located approximately 500 bp downstream ofthe Phi33 insertion site that is within the intergenic regionimmediately downstream of the tail fibre operon (FIG. 6 ). Primer B4901consists of 5′ AvrII and XhoI restriction sites (underlined), followedby sequence of Phi33 that is complementary to sequence locatedimmediately downstream of the Phi33 insertion site (FIG. 6 ). PrimerB4902 consists of a 5′ AvrII restriction site (underlined), followed byPhi33 sequence located immediately upstream of the insertion site (FIG.6 ). Primer B4903 consists of a 5′ NheI site (underlined), followed byPhi33 sequence that is complementary to sequence located approximately500 bp upstream of the Phi33 insertion site (FIG. 6 ).

Primer B4900 (SEQ ID NO: 31) 5′ gatagctagcTTTCTCGTTTTAATGTCG 3′Primer B4901 (SEQ ID NO: 32) 5′ gataCCTAGGtgCTCGAGTATTCGCCCAAAAGAAAAG 3′Primer B4902 (SEQ ID NO: 33) 5′ gataCCTAGGTCAGGAGCCTTGATTGATC 3′Primer B4903 (SEQ ID NO: 34) 5′ gatagctagcGGACTGGTAAGTCTGGTG 3′

2. Plasmid pSMX252 (FIG. 6 ), comprising pSMX251 containing SASP-C underthe control of an rpsB promoter, may be constructed as follows.

The SASP-C gene from Bacillus megaterium strain KM (ATCC 13632) may beamplified by PCR using primers B4904 and B4270 (FIG. 6 ). The resultingPCR product may then be digested with XhoI and NcoI. The rpsB promotermay be amplified by PCR from P. aeruginosa using primers B4271 and B4905(FIG. 6 ). The resulting PCR product may then be digested with NcoI andAvrII. The two digested PCR products may then be cleaned and ligated topSMX251 that has been digested with XhoI and AvrII, yielding plasmidpSMX252 (FIG. 6 ).

Primer B4904 comprises a 5′ XhoI restriction site, followed by 5 bases,and then a bi-directional transcriptional terminator, and then sequencecomplementary to the 3′ end of the SASP-C gene from B. megaterium strainKM (ATCC 13632) (underlined, in bold; FIG. 6 ). Primer B4270 comprises a5′ NcoI restriction site (underlined), followed by sequence of the 5′end of the SASP-C gene from B. megaterium strain KM (ATCC 13632) (FIG. 6). Primer B4271 comprises a 5′ NcoI restriction site (underlined),followed by sequence complementary to the end of the rpsB promoter fromP. aeruginosa PA01 (FIG. 6 ). Primer B4905 comprises a 5′ AvrIIrestriction site (underlined), followed by sequence of the beginning ofthe rpsB promoter from P. aeruginosa PAO1 (FIG. 6 ).

Primer B4904 (SEQ ID NO: 35)5′-gataCTCGAGGATCTAGTCAAAAGCCTCCGACCGGAGGCTTTTGACT ttagtacttgccgcctag-3′Primer B4270 (SEQ ID NO: 36) 5′- gataccATGGcaaattatcaaaacgcatc-3′Primer B4271 (SEQ ID NO: 37) 5′-gataCCATggTAGTTCCTCGATAAGTCG-3′Primer B4905 (SEQ ID NO: 38)5′-gataCCTAGGCCTAGGgatctGACCGACCGATCTACTCC-3′

3. pSMX253 (FIG. 6 ), comprising pSMX252 containing lacZα, may beconstructed as follows.

lacZα may be PCR amplified using primers B4906 and B4907 (FIG. 6 ). Theresulting PCR product may then be digested with XhoI and ligated topSMX252 that has also been digested with XhoI and treated with alkalinephosphatase prior to ligation, to yield pSMX253 (FIG. 6 ).

Primer B4906 consists of a 5′ XhoI restriction site (underlined),followed by sequence complementary to the 3′ end of lacZα (FIG. 6 ).Primer B4907 consists of a 5′ XhoI restriction site (underlined),followed by sequence of the lac promoter driving expression of lacZα(FIG. 6 ).

Primer B4906 (SEQ ID NO: 39) 5′-gataCTCGAGttagcgccattcgccattc-3′Primer B4907 (SEQ ID NO: 40) 5′-gataCTCGAGgcgcaacgcaattaatgtg-3′Genetic Modification of Phi33, PTPX83, PTPX84, and Similar Phage, toIntroduce rpsB-SASP-C and lacZα

-   -   1. Plasmid pSMX253 (FIG. 6 ; FIG. 3 ; FIG. 4 ; FIG. 7 ) may be        introduced into P. aeruginosa strain PAX21 by conjugation,        selecting transconjugants on the basis of tetracycline        resistance (50 μg/ml), yielding strain PTA51.    -   2. Strain PTA51 may be infected in individual experiments with        phage Phi33, or PTPX83, or PTPX84, or other similar phage, and        the progeny phage harvested.    -   3. Recombinant phage, in which rpsB-SASP-C and lacZα have been        introduced into the chosen insertion site, may be identified by        plaquing the lysate from step (2) on P. aeruginosa strain PAX21,        onto medium containing S-gal, looking for black plaques, which        are indicative of β-galactosidase activity.    -   4. PCR may be carried out to check that rpsB-SASP-C and lacZα        are present.    -   5. Following identification of verified isolates (for example,        PTPX85 (FIG. 7 ), PTPX86 (FIG. 3 ), PTPX87 (FIG. 4 )), the        isolates may be plaque purified twice more on P. aeruginosa        strain PAX21, prior to further use.        Genetic Modification to Remove the lacZα Marker from PTPX85,        PTPX86, PTPX87, and Similar Derivatives of Phi33    -   1. Plasmid pSMX252 (FIG. 6 ; FIG. 3 ; FIG. 4 ; FIG. 7 ) may be        introduced into P. aeruginosa strain PAX21 by conjugation,        selecting transconjugants on the basis of tetracycline        resistance (50 μg/ml), yielding strain PTA85.    -   2. Strain PTA85 may be infected in individual experiments with        phage PTPX85, or PTPX86, or PTPX87, or other similar phage, and        the progeny phage harvested.    -   3. Recombinant phage, in which lacZα marker has been removed,        may be identified by plaquing the lysate from step (2) on P.        aeruginosa strain PAX21, onto medium containing S-gal, looking        for white plaques, which are indicative of loss of        β-galactosidase activity.    -   4. PCR may be carried out to confirm removal of the lacZα        marker, while ensuring that rpsB-SASP-C is still present.    -   5. Following identification of verified isolates (for example,        PTPX88 (FIG. 7 ), PTPX89 (FIG. 3 ), PTPX90 (FIG. 4 )), the        isolates may be plaque purified twice more on P. aeruginosa        strain PAX21, prior to further use.

Construction of a plasmid to introduce fda-SASP-C (codon optimised) andlacZα into a chosen insertion site, located in an intergenic regionimmediately downstream of the tail fibre operon, within the genome ofPhi33, PTPX83, PTPX84, and similar phage

1. Plasmid pSMX254 (FIG. 8 ), comprising pSMX251 containing SASP-C codonoptimised for expression in P. aeruginosa, under the control of an fdapromoter, may be constructed as follows.

The SASP-C gene from Bacillus megaterium strain KM (ATCC 13632) may becodon optimised for expression in P. aeruginosa (FIG. 9 ) andsynthesised in vitro. The codon optimised SASP-C gene may then beamplified by PCR using primers B4312 and B4313 (FIG. 8 ). The fdapromoter may be amplified by PCR from P. aeruginosa using primers B4314and B4315 (FIG. 8 ). The resulting two PCR products may then be joinedby splicing by overlap extension (SOEing) PCR, using the outer primersB4312 and B4314 (FIG. 8 ). The resulting fda-codon optimisedSASP-C-terminator PCR product may then be digested with XhoI and AvrII,cleaned, and ligated to pSMX251 that has been digested with XhoI andAvrII, yielding plasmid pSMX254 (FIG. 8 ).

Primer B4312 comprises a 5′ XhoI restriction site, followed by abi-directional transcriptional terminator, and then sequencecomplementary to the 3′ end of the SASP-C gene from B. megaterium strainKM (ATCC 13632) that has been codon optimised for expression in P.aeruginosa (underlined, in bold; FIG. 8 ). Primer B4313 comprisessequence of the 3′ end of the fda promoter from P. aeruginosa PA01 (inbold) followed by sequence of the 5′ end of the codon optimised SASP-Cgene. Primer B4314 comprises sequence complementary to the 5′ end of thecodon optimised SASP-C gene followed by sequence complementary to the 3′end of the fda promoter from P. aeruginosa PAO1 (FIG. 8 ). Primer B4315comprises a 5′ AvrII restriction site (underlined), followed by sequenceof the beginning of the fda promoter from P. aeruginosa PAO1 (FIG. 8 ).

Primer B4312 (SEQ ID NO: 41)5′-gataCTCGAGAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTTCAGT ACTTGCCGCCCAG-3′Primer B4313 (SEQ ID NO: 42)5′- GATTGGGAGATACGAGAACCATGGCCAACTACCAGAACGC -3′ Primer B4314(SEQ ID NO: 43) 5′- GCGTTCTGGTAGTTGGCCATGGTTCTCGTATCTCCCAATC-3′Primer B4315 (SEQ ID NO: 44) 5′- GATACCTAGGAACGACGAAGGCCTGGTG-3′

3. pSMX255 (FIG. 8 ), comprising pSMX254 containing lacZα, may beconstructed as follows.

lacZα may be PCR amplified using primers B4906 and B4907 (FIG. 8 ). Theresulting PCR product may then be digested with XhoI and ligated topSMX254 that has also been digested with XhoI and treated with alkalinephosphatase prior to ligation, to yield pSMX255 (FIG. 8 ).

Primer B4906 consists of a 5′ XhoI restriction site (underlined),followed by sequence complementary to the 3′ end of lacZα (FIG. 8 ).Primer B4907 consists of a 5′ XhoI restriction site (underlined),followed by sequence of the lac promoter driving expression of lacZα(FIG. 8 ).

Primer B4906 (SEQ ID NO: 40) 5′-gataCTCGAGttagcgccattcgccattc-3′Primer B4907 (SEQ ID NO: 41) 5′-gataCTCGAGgcgcaacgcaattaatgtg-3′Genetic Modification of Phi33, PTPX83, PTPX84, and Similar Phage, toIntroduce Fda-Codon Optimised SASP-C and lacZα

-   -   1. Plasmid pSMX255 (FIG. 8 ; FIG. 10 ; FIG. 11 ; FIG. 12 ) may        be introduced into P. aeruginosa strain PAX21 by conjugation,        selecting transconjugants on the basis of tetracycline        resistance (50 μg/ml), yielding strain PTA86.    -   2. Strain PTA86 may be infected in individual experiments with        phage Phi33, or PTPX83, or PTPX84, or other similar phage, and        the progeny phage harvested.    -   3. Recombinant phage, into which fda-codon optimised SASP-C and        lacZα have been introduced, may be identified by plaquing the        lysate from step (2) on P. aeruginosa strain PAX21, onto medium        containing S-gal, looking for black plaques, which are        indicative of β-galactosidase activity.    -   4. PCR may be carried out to check that fda-codon optimised        SASP-C and lacZα are present.    -   5. Following identification of verified isolates (for example,        PTPX91 (FIG. 12 ), PTPX92 (FIG. 10 ), PTPX93 (FIG. 11 )), the        isolates may be plaque purified twice more on P. aeruginosa        strain PAX21, prior to further use.        Genetic Modification to Remove the lacZα Marker from PTPX91,        PTPX92, PTPX93, and Similar Derivatives of Phi33    -   1. Plasmid pSMX254 (FIG. 8 ; FIG. 10 ; FIG. 11 ; FIG. 12 ) may        be introduced into P. aeruginosa strain PAX21 by conjugation,        selecting transconjugants on the basis of tetracycline        resistance (50 μg/ml), yielding strain PTA87.    -   2. Strain PTA87 may be infected in individual experiments with        phage PTPX91, or PTPX92, or PTPX93, or other similar phage, and        the progeny phage harvested.    -   3. Recombinant phage, in which lacZα marker has been removed,        may be identified by plaquing the lysate from step (2) on P.        aeruginosa strain PAX21, onto medium containing S-gal, looking        for white plaques, which are indicative of loss of        β-galactosidase activity.    -   4. PCR may be carried out to confirm removal of the lacZα        marker, while ensuring that fda-codon optimised SASP-C is still        present.    -   5. Following identification of verified isolates (for example,        PTP387 (FIG. 12 ), PTP388 (FIG. 10 ), PTP389 (FIG. 11 )), the        isolates may be plaque purified twice more on P. aeruginosa        strain PAX21, prior to further use.

REFERENCES

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1. A modified bacteriophage capable of infecting a plurality ofdifferent target bacteria, which bacteriophage includes a toxin geneencoding a toxin protein which is toxic to the target bacteria; whereinthe bacteriophage is lytic; and wherein the bacteriophage expresses hostrange determinant proteins which have a plurality of bacterial hostspecificities. 2-45. (canceled)
 46. A modified bacteriophage capable ofinfecting a plurality of different target bacteria, which bacteriophagecomprises a variant of bacteriophage Phi33 which has been modified tocomprise an α/β small acid-soluble spore protein (SASP) gene encoding aSASP gene which is expressed under the control of a constitutivepromoter, and further wherein the bacteriophage is lytic.
 47. A modifiedbacteriophage according to claim 46, wherein the tail fiber proteincomprises a receptor binding region that binds to the target bacteriumand a region linking the receptor binding region to the body of thebacteriophage.
 48. A modified bacteriophage according to claim 46,wherein the constitutive promoter is selected from pdhA, rspB, pgi, fda,and lasB.
 49. A modified bacteriophage according to claim 46, whereinthe constitutive promoter is fda.
 50. A modified bacteriophage accordingto claim 46, wherein the SASP is SASP-C.
 51. A modified bacteriophageaccording to claim 50, wherein the SASP-C is from Bacillus megaterium.52. A modified bacteriophage according to claim 46, wherein the targetbacteria includes Pseudomonas.
 53. A modified bacteriophage according toclaim 46, wherein the target bacteria includes Pseudomonas aeruginosa.54. A composition comprising a modified bacteriophage according to claim46, and a pharmaceutically acceptable carrier.
 55. A compositioncomprising a modified lytic bacteriophage according to claim 46 inadmixture with at least one other modified lytic bacteriophage which iscapable of infecting target bacteria, which also includes a SASP geneencoding a SASP which is toxic to a target bacteria.
 56. A compositionaccording to claim 55, which comprises at least two of said modifiedlytic bacteriophages, wherein at least two of which have different hostspecificities.
 57. A modified bacteriophage according to claim 46, whichcomprises a SASP-C gene comprised of P. aeruginosa optimized codons,which gene is operably linked to the fda promoter.
 58. A modifiedbacteriophage according to claim 46, which consists of a variant ofbacteriophage Phi33 wherein the modifications consist of theincorporation of a SASP-C gene which is comprised of P. aeruginosaoptimized codons, and which gene is operably linked to the fda promoter,and further wherein the bacteriophage is lytic.
 59. A compositionaccording to claim 54 which is formulated for pharmaceutical use, fortopical use, or for delivery to the respiratory tract.
 60. A compositionaccording to claim 58 which is formulated for pharmaceutical use, fortopical use, or for delivery to the respiratory tract.
 61. A method ofkilling target bacteria comprising contacting said target bacteria witha bactericidally effective amount of at least one modified bacteriophageaccording to claim 46, which is toxic to the target bacteria.
 62. Amethod of killing target bacteria comprising contacting said targetbacteria with a bactericidally effective amount of at least one modifiedbacteriophage according to claim 58, which is toxic to the targetbacteria.
 63. The method of claim 61, which is effected in vitro. or invivo, by administering a bactericidally effective amount of saidmodified bacteriophage to a subject infected by said target bacterium.64. The method of claim 62, which is effected in vitro. or in vivo, byadministering a bactericidally effective amount of said modifiedbacteriophage to a subject infected by said target bacterium.